CN111693518B - Mercury ion detection method - Google Patents
Mercury ion detection method Download PDFInfo
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- CN111693518B CN111693518B CN201910191366.4A CN201910191366A CN111693518B CN 111693518 B CN111693518 B CN 111693518B CN 201910191366 A CN201910191366 A CN 201910191366A CN 111693518 B CN111693518 B CN 111693518B
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- 238000001514 detection method Methods 0.000 title abstract description 22
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 title abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 24
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical group CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims abstract description 12
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229940025294 hemin Drugs 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 34
- 239000000446 fuel Substances 0.000 claims description 15
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 14
- 229910052753 mercury Inorganic materials 0.000 claims description 12
- -1 mercury ions Chemical class 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000002371 ultraviolet--visible spectrum Methods 0.000 claims description 5
- 101001128634 Homo sapiens NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrial Proteins 0.000 claims description 3
- 102100032194 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2, mitochondrial Human genes 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 12
- 229940113082 thymine Drugs 0.000 abstract description 10
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- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 6
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 4
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- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 229910004298 SiO 2 Inorganic materials 0.000 description 1
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- ASJWEHCPLGMOJE-LJMGSBPFSA-N ac1l3rvh Chemical compound N1C(=O)NC(=O)[C@@]2(C)[C@@]3(C)C(=O)NC(=O)N[C@H]3[C@H]21 ASJWEHCPLGMOJE-LJMGSBPFSA-N 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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Abstract
The invention provides a preparation method of thymine-Hg 2+ -thymine (T-Hg) 2+ -T) mercury ion detection method combining coordination chemistry and entropy-driven catalytic color reaction. Catalytic reaction driven by entropy is T-Hg 2+ -T coordination is chemically induced resulting in the release of G-rich sequences. Can form a hemin/G-tetrad structure with the help of hemin to form a DNA mimic enzyme with catalase activity, oxidize a substrate 3,3',5,5' -tetramethylbenzidine, change the color from colorless to blue, and have a good linear relationship between 0.05nM and 2 nM. The method can be used for Hg in water 2+ On-site analysis of concentration.
Description
Technical Field
The invention relates to the field of detection of mercury ions, in particular to thymine-Hg 2+ -thymine (T-Hg) 2+ -T) coordination chemistry and entropy-driven catalytic color reaction.
Background
Mercury pollution has become a global problem for human health and environmental protection. Although mercury naturally occurs in the environment, human activities cause mercury to enter the environment, such as mining, fossil fuel combustion, and solid waste incineration. Trace amount of Hg 2+ May accumulate in the human body by biological accumulation through binding to thiol-containing molecules. Hg in the human body 2+ Even at very low concentrations, neurological, renal and other diseases can be caused due to its high toxicity. Therefore, there is a need to develop a method for detecting Hg in the fields of food safety and environmental protection 2+ The method of (1). Colorimetric methods have attracted much attention because the naked eye can easily, quickly, and observe results. Different kinds of colorimetric methods have been used based on gold nanoparticles, graphene, SiO 2 Hg of nanoparticles, silver nanoparticles, G-tetrads, and the like 2+ And (6) detecting. However, the above methods have insufficient sensitivity or poor selectivity.
The prior art has proved that thymine-thymine mismatched base pairs and Hg 2+ High affinity in between. thymine-Hg 2+ -thymine (T-Hg) 2+ -T) coordination chemistry has been combined with colorimetry, fluorescence, electrochemistry, electrochemiluminescence and enhanced Raman scattering. However, the detection limit of the prior art is higher than 30nM (the maximum pollutant level of mercury in drinking water set by the world health organization), and cannot meet the requirements of practical application. In order to obviously improve the sensitivity, enzyme-free signal enhancement methods such as hybridization chain reaction, hairpin catalytic self-assembly, catalytic self-assembly and the like are used for thymine-Hg in the prior art 2+ -thymine (T-Hg) 2+ -T) coordinated Hg 2+ And (6) detecting. Most of these methods are driven by the free energy of base pair formation, which can lead to relatively high background and false positive results.
Thymine cannot be obtainedpyridine-Hg 2+ -thymine (T-Hg) 2+ -T) report of a mercury ion detection method combining coordination chemistry and entropy-driven catalytic color reaction.
Disclosure of Invention
In order to solve the problems, the invention provides a method for dissolving thymine-Hg 2+ -thymine (T-Hg) 2+ -T) mercury ion detection method combining coordination chemistry and entropy-driven catalytic color reaction.
The invention comprises the following steps:
(1) adding a proper amount of DNA compound, a sequence T and a fuel chain F into a solution to be detected, wherein the sequence T is as follows: 5 '-TTCATTGTGTTACTTT _ tctctcca-3'; the DNA complex is formed by hybridizing a sequence R, a sequence Q and a sequence P, wherein the sequence R is 5'-GGGTTGGGCGGGATGGGTTTCAC-3', the sequence Q is 5 '-TGGAGA _ TTTGTTTCTCTTTGTT _ AGGG _ GTGAAACCCATCCCG-3', and the sequence P is 5 '-CCACATACATCATATT _ CCCT _ AACAAAGAGAAACAAA-3'; the fuel train F is 5 '-CGGGATGGGTTTCAC _ CCCT _ AACAAAGAGAAACAAA-3';
(2) adding the solution obtained in the step (1) into MgCl containing a proper amount 2 Incubating in Tris-HCl buffer solution;
(3) adding a proper amount of hemin and a proper amount of KCl into the solution obtained in the step (2);
(4) adding the solution obtained in the step (3) into a solution containing appropriate amounts of 3,3',5,5' -tetramethylbenzidine and H 2 O 2 In Tris-HCl solution;
(5) the UV-vis absorption spectrum was measured and the concentration of mercury ions in the solution was calculated using a standard curve method.
Preferably, the DNA complex formation step is as follows: 50nM sequence P, 50nM sequence R and 50nM sequence Q were mixed together and heated to 90 ℃ in 10mM Tris-HCl solution (pH8) for 5 minutes, then slowly cooled to form DNA complexes.
Preferably, the DNA complex, sequence T and fuel chain F added in step (1) are 50nM, 100nM and 100nM, respectively.
Preferably, the step (2) is specifically to add the solution obtained in the step (1) to a solution containing 5mM MgCl 2 In 10mM Tris-HCl buffer solution (pH 7.5) at 37 ℃ for 120 minutes.
Preferably, the hemin and KCl added in step (3) are 30nM and 25mM, respectively.
Preferably, the step (4) is specifically: adding the solution obtained in step (4) to a solution containing 600. mu.M of 3,3',5,5' -tetramethylbenzidine and 10mM of H 2 O 2 In Tris-HCl solution (pH 5.5).
The invention creatively uses thymine-Hg 2+ -thymine (T-Hg) 2+ -T) coordination chemistry and entropy-driven catalytic color reaction are combined, so that mercury ion signals are amplified in multiple stages, the sensitivity is improved, the signals appear in a visual form, the detection is intuitive and easy, and the cost is reduced.
Drawings
FIG. 1(A) different concentrations of Hg 2+ UV-vis absorption spectrum curves (0.05nM, 0.1nM, 1nM,2nM,4nM,5nM and 10 nM). (B) As Hg 2+ Delta absorbance intensity as a function of concentration and Hg with different concentrations 2+ A photograph of the sample of (1). Illustration is shown: the concentration calibration curve was in the range of 0.05nM to 2 pM.
FIG. 2 is a schematic diagram of the detection process of the present invention.
FIG. 3 shows UV-visible absorption spectrum curves and delta absorbance intensities for different samples of comparative example one (inset: photo).
FIG. 4 is a comparison of the absorbance of different samples in comparative example II.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in further detail with reference to examples.
Example 1
Drawing a standard curve: preparing a mercury ion standard solution with gradient concentration, wherein the solution concentration is respectively as follows: 0.05nM,0.1nM,0.5nM,1nM,2nM,4nM,5nM,10 nM. An appropriate amount of the above standard solution was mixed with 100nM of the sequence T, 50nM of the DNA complex and 100nM of the fuel chain F, respectively, at 37 ℃ in the presence of 5mM MgCl 2 In 10mM Tris-HCl buffer solution (pH 7.5) for 120 minutes. Then, 30nM hemin and 25mM KCl were added to the above solution, and finally, 600. mu.M 3,3',5,5' -tetramethylbenzidine and 10mM H were added 2 O 2 In Tris-HCl solution (pH 5.5)In a color reaction. The UV-vis absorption spectrum was detected by a UV-vis spectrophotometer. As shown in FIG. 1A, the absorbance intensity is a function of Hg 2+ The concentration increases. Absorbance intensity and Hg at 0.05nM to 2nM 2+ A good linear correlation between concentrations was shown (fig. 1B). The regression equation can be expressed as Y-0.11331 +0.0575C (R) 2 0.999), wherein Y and C represent absorbance intensity and Hg, respectively 2+ And (4) concentration. Limit of detection (LOD) was calculated as 7pM, Hg 2+ The limit of visual detection of (D) was about 0.1 nM. And drawing an absorbance-concentration standard curve.
An appropriate amount of the test solution was mixed with 100nM of sequence T, 50nM DNA complex and 100nM of fuel chain F at 37 ℃ in the presence of 5mM MgCl 2 In 10mM Tris-HCl buffer solution (pH 7.5) for 120 minutes. Then, 30nM hemin and 25mM KCl were added to the above solution, and finally, 600. mu.M 3,3',5,5' -tetramethylbenzidine and 10mM H were added 2 O 2 In Tris-HCl solution (pH 5.5) for color reaction. The UV-vis absorption spectrum was detected by a UV-vis spectrophotometer. And calculating the concentration of mercury ions in the solution to be detected through a standard curve.
The detection microscopic process is shown in FIG. 2: sequence Q is shown as chain 1, 2, 3, 4; sequence R is shown as chain 1; sequence P is shown as chain 5, 2, 3; sequence T is shown as chain 3', 4; the fuel chain F is shown as chain 1 ", 2", 3 ". The rear end of sequence T (denoted 4) may be joined to the front end of sequence Q (denoted 4). A thymine-rich portion is present in the tail of sequence Q. In Hg 2+ T-Hg between sequences T and Q in the presence of 2+ -T coordination chemistry can replace sequence R from the DNA complex and then expose domain 2 of sequence Q. Domain 2 then becomes the foothold of the fuel chain F displacement sequences R and T. The sequence T can enter the next cycle and cause the release of a large number of sequences R. The hemin and the G-rich sequence R form a hemin/G-tetrad structure to form a DNA mimic enzyme with catalase activity, and the substrate 3,3',5,5' -tetramethyl benzidine is oxidized, and the color is changed from colorless to blue.
Example 2
Water samples were collected from tap water, streams and spring water. Prior to the measurement of the measurement,all water samples were filtered through 0.22 μm membranes to remove solid residues. The detection process is the same as that of example 1, the detection results are shown in Table 1, and Hg in the actual water sample 2+ The concentrations of the water are tap water, brook water and spring water respectively. The recovery rate increased from 91.6% to 105.3%. These results show that the detection of mercury ions in real environmental water samples has great prospects.
TABLE 1 true water sample test results
Comparative example 1
In order to embody the technical effects of the above examples, the following comparative examples are provided: 1. the blank sample, the solution to be tested does not contain mercury ions, and the rest of the detection process is the same as that of example 1. 2. The sequence T in example 1 was replaced by the sequence T', which is: 5 '-CCCACCGCGCCACCCC _ TCTCCA-3', the rest of the detection process is the same as in example 1. 3. The test procedure was the same as in example 1, except that no fuel train F was added. 4. The concentration of fuel chain F was half that of example 1 and the rest of the test procedure was the same as example 1. 5. The DNA complex is formed by sequences P, R 'and Q', sequence R '5'-CTACGTCTCCAACTAACTTACGG-3', sequence Q' being: 5 '-TGGAGA _ TTTGTTTCTCTTTGTT _ AGGG _ CCGTAAGTTAGTTGGAGACGTAG-3', the rest of the detection process is the same as in example 1. 6. The procedure was as in example 1.
The results are shown in FIG. 3, comparative example 1, where the sample contained no Hg 2+ And the detection result shows the intensity of the background absorbance. In comparative example 2, a low absorbance intensity was obtained due to the replacement of the thymine region of sequence T. In comparative example 3, the entropy-driven catalytic reaction could not proceed without a fuel chain, showing a weak absorption intensity demonstrating T-Hg 2+ It is important that the-T base pair initiates an entropy-driven catalytic reaction. In comparative example 4, the sample with half concentration of fuel chains showed strong absorption intensity and light blue color, demonstrating that the entropy-driven catalytic reaction cannot be completely completed with only half concentration of fuel chains. In comparative example 5, the G-rich region of sequence R was replaced with R' without the G-rich region, resulting inResulting in a weak absorbance intensity. The hemin/G-quadruplex structure cannot be formed because of the absence of a G-rich region. The detection result of sample 6 showed strong absorption intensity and deep blue color, and all reaction components showed T-Hg 2+ T-coordination chemistry successfully initiated the entropy-driven catalytic reaction and successfully formed the hemin/G-quadruplex structure, forming a DNA mimic enzyme with catalase activity, oxidizing the substrate 3,3',5,5' -tetramethylbenzidine, changing color from colorless to blue.
Comparative example No. two
The mercury ions in example 1 were replaced with Cu of the same concentration, respectively 2+ ，Ca 2+ ，Pb 2+ ，Co 2+ ，Fe 2+ ，Sn 2+ And Zn 2 + The rest of the detection process was the same as in example 1. The results of the detection are shown in FIG. 4, which shows Hg in the sample 2+ Showing a strong response signal. However, the signal obtained with other interfering metal ions is negligible. And 2nM Hg 2+ The signal strength of interfering metal ions is much weaker than the signal. Due to T-Hg 2+ The strong affinity of the T coordination chemistry, these results demonstrate the good selectivity of the proposed method.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (6)
1. A method for detecting the concentration of mercury ions in a solution comprises the following steps:
(1) adding a proper amount of DNA compound, a sequence T and a fuel chain F into a solution to be detected, wherein the sequence T is as follows: 5 '-TTCATTGTGTTACTTT _ tctctcca-3'; the DNA complex is formed by hybridizing a sequence R, a sequence Q and a sequence P, wherein the sequence R is 5'-GGGTTGGGCGGGATGGGTTTCAC-3', the sequence Q is 5 '-TGGAGA _ TTTGTTTCTCTTTGTT _ AGGG _ GTGAAACCCATCCCGCCCAACCC-3', and the sequence P is 5 '-CCACATACATCATATT _ CCCT _ AACAAAGAGAAACAAA-3'; the fuel train F is 5 '-GGGTTGGGCGGGATGGGTTTCAC _ CCCT _ AACAAAGAGAAACAAA-3';
(2) adding the solution obtained in the step (1) into MgCl containing a proper amount 2 Is incubated in Tris-HCl buffer solution;
(3) adding a proper amount of hemin and a proper amount of KCl into the solution obtained in the step (2);
(4) adding the solution obtained in the step (3) into a solution containing appropriate amounts of 3,3',5,5' -tetramethylbenzidine and H 2 O 2 In Tris-HCl solution;
(5) the UV-vis absorption spectrum was measured and the concentration of mercury ions in the solution was calculated using a standard curve method.
2. The method of claim 1, wherein said DNA complex forming step comprises: 50nM sequence P, 50nM sequence R and 50nM sequence Q are mixed together and heated to 90 ℃ for 5 minutes in 10mM Tris-HCl solution at pH8, then slowly cooled to form DNA complexes.
3. The method according to any of the preceding claims, wherein the DNA complex, the sequence T and the fuel strand F are added in step (1) at 50nM, 100nM and 100nM, respectively.
4. The method according to claim 3, wherein step (2) comprises adding the solution obtained in step (1) to a solution containing 5mM MgCl at pH 7.5 2 Was incubated at 37 ℃ for 120 minutes in 10mM Tris-HCl buffer.
5. The method according to claim 4, wherein the hemin and KCl are added in step (3) at 30nM and 25mM, respectively.
6. The method according to claim 5, wherein the step (4) is embodied as: adding the solution obtained in step (4) to a solution containing 600. mu.M of 3,3',5,5' -tetramethylbenzidine and 10mM of H 2 O 2 In a Tris-HCl solution at a pH of 5.5.
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